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Objective:To investigate the inhibitory effect and killing mechanism of Bcl-2 BH4 selective inhibitor BDA-366 on NK/T cell lymphoma (NK/TCL) .Methods:Human NK cell leukemia cell line YT and human NK/TCL cell line NK92 cells were treated with 0, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50 μmol/L BDA-366. CCK-8 assay was used to calculate the half inhibitory concentration (IC 50) value of BDA-366 on these cells. The apoptosis levels of cells in control group and IC 50 BDA-366 treated group were detected by flow cytometry. Western blotting was used to detect the expression levels of apoptosis-related proteins in cells of control group and 1/2 IC 50, IC 50, 2× IC 50 BDA-366 treated groups. TMRE and Fluo-3 fluorescent probe were used to detect mitochondrial membrane potential of control group and IC 50 BDA-366 treated group, and the intracellular Ca 2+ concentration of control group, IC 50, 2× IC 50 BDA-366 treated groups. NOD-SCID mice in control group and 10 mg/kg BDA-366 intraperitoneal injection group were weighed and HE staining was performed to evaluate the toxicity of BDA-366 in vivo. Results:The IC 50 of BDA-366 for YT and NK92 cells were 0.065 and 0.086 μmol/L respectively. The apoptosis rates of YT cells in the control group and 0.065 μmol/L BDA-366 group were (6.62±1.59) % and (34.60±3.06) % respectively. The apoptosis rates of NK92 cells in the control group and 0.086 μmol/L BDA-366 group were (5.57±0.88) % and (29.18±0.90) % respectively, both with statistically significant differences ( t=14.05, P<0.001; t=32.58, P<0.001). The relative expression of Bax in NK92 cells of the control group, 0.043, 0.086 and 0.172 μmol/L BDA-366 groups were 0.85±0.00, 1.26±0.04, 1.51±0.18, 1.15±0.10 ( F=20.70, P<0.001), the relative expression of Bax in BDA-366 groups were higher than that in the control group (all P<0.05). The fluorescence intensity of TMRE of YT cells in the control group and 0.065 μmol/L BDA-366 group were 8 372.00±330.47 and 6 419.67±311.34, and that of NK92 cells in the control group and 0.086 μmol/L BDA-366 group were 9 169.00±535.72 and 7 311.67±295.52 respectively, and there were statistically significant differences ( t=7.45, P=0.002; t=5.26, P=0.006). In YT cells, the intracellular Ca 2+ concentrations of 0.065 and 0.130 μmol/L BDA-366 groups were significantly higher than that of the control group (5 791.67±220.45, 6 729.33±585.39, 4 874.67±112.61, F=19.16, P=0.003) ( P=0.039; P=0.002). In NK92 cells, the intracellular Ca 2+ concentrations of 0.086 and 0.172 μmol/L BDA-366 groups were significantly higher than that of the control group (4 553.67±17.62, 4 740.33±254.50, 4 185.67±17.67, F=10.96, P=0.010) ( P=0.039; P=0.007). There was no statistically significant difference in body weight change on day 12 compared with day 0 of NOD-SCID mice between BDA-366 group and control group [ (3.18±0.01) g vs. (2.73±0.58) g, t=0.60, P=0.570], and HE staining showed no abnormal morphology of heart, liver, spleen, lung and kidney in BDA-366 group. Conclusion:BDA-366 promotes NK/TCL cells apoptosis in vitro, but does not cause weight loss and morphological changes of organs by HE staining in vivo. The inhibitory effect of BDA-366 on NK/TCL cells may be achieved by increasing Bax expression, inducing Ca 2+ release and reducing mitochondrial membrane potential.
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Objective To explore the molecular mechanism underlying the anti-apoptotic activity of pORF5 plasmid protein of Chlamydia trachomatis,so as to provide an experimental basis for further clarifying the pathogenesis of Chlamydia trachomatis.Methods HeLa cells were divided into two groups:carbonyl cyanide m-chlorophenyl hydrazone (CCCP,an apoptosis inducer) group was stimulated by CCCP for 30 minutes,and pORF5 + CCCP group was pretreated with pORF5 plasmid protein for 18 hours followed by CCCP for 30 minutes.Then,Western blot analysis was performed to determine the expression of apoptosisrelated proteins Bcl-2,Bax and caspase-3,JC-1 fluorescent probe was used to detect changes in the mitochondrial membrane potential in HeLa cells,and cytochrome c release from mitochondria was analyzed by indirect immunofluorescence assay.To analyze whether high-mobility group box 1 (HMGB1) protein participated in the anti-apoptotic role of pORF5 plasmid protein,HMGB 1 shRNA and control RNA were separately transfected into the HeLa cells,which were then stimulated by pORF5 plasmid protein and CCCP.Then,the protein expression of Bcl-2,Bax,activated caspase-3 was determined,and cytochrome c release was analyzed.Data were compared between two groups by using paired t test.Results pORF5 plasmid protein could antagonize the CCCP-induced decrease of mitochondrial membrane potential,and the red/green fluorescence intensity ratio was significantly lower in the CCCP group (0.4 ± 0.1) than in the pORF5 + CCCP group (1.7 ± 0.3;t =6.95,P ＜ 0.01).The protein expression of Bcl-2 in the HeLa cells in the pORF5 + CCCP group was 5.3 ± 0.6 times more than that in the CCCP group (t =8.62,P ＜ 0.01),while the protein expression of Bax and activated caspase-3 in the pORF5 + CCCP group significantly decreased by 79％ ± 10％ (t =9.23,P ＜ 0.01) and 75％ ± 8％ (t =4.26,P ＜ 0.05) respectively compared with the CCCP group.Compared with the control RNA transfection group,the HMGB1 shRNA transfection group showed significantly decreased mitochondrial membrane potential in the HeLa cells (t =11.23,P ＜ 0.01),increased cytochrome c release,decreased Bcl-2 expresson (t =7.19,P ＜ 0.05) and increased Bax expression (t =13.06,P ＜ 0.01) after stimulation with pORF5 and CCCP.Conclusion Chlamydia trachomatis plasmid protein pORF5 plays an anti-apoptosis role by blocking the mitochondrial apoptotic pathway through HMGB1 protein.
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High-grade B-cell lymphomas with rearrangement of MYC, BCL-2 and/or BCL-6 were introduced by the update of the WHO classification of lymphoid neoplasms. They usually present unique morphological and molecular characteristics, with an aggressive clinical outcome and worse prognosis. We report a 48 year-old female patient presenting with B symptoms and enlarged lymph nodes. Blood count showed pancytopenia and peripheral blood smears showed large lymphoid cells, some with nuclei and vacuoles. LDH was 3524 g/L and serum calcium was 11.5 mg/dL. Flow cytometry immunophenotyping showed pathological mature B lymphocytes. Protein electrophoresis showed a slight monoclonal peak. The biopsy disclosed a triple expressor diffuse large B-cell lymphoma, arising from germinal center. FISH was positive for MYC, BCL-2 and BCL-6 (triple hit) with a clonal evolution. Conventional cytogenetics showed a complex karyotype. Chemotherapy was started with R-CHOP (Rituximab/cyclophosphamide/doxorubicin/vincristine/prednisone). She developed impaired consciousness; the brain CT scan showed a large brain mass. The patient died within 3 weeks.
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Humans , Female , Middle Aged , Translocation, Genetic/genetics , Lymphoma, Large B-Cell, Diffuse/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-6/genetics , Hypercalcemia/etiology , Tomography, X-Ray Computed , Lymphoma, Large B-Cell, Diffuse/complications , Lymphoma, Large B-Cell, Diffuse/pathology , Fatal Outcome , KaryotypeABSTRACT
Objective To explore the effects of delayed mild hypothermia (MHT) in different time windows on the expressions of Bcl-2, Bax and Caspase-3 in brain tissue of model rats with traumatic brain injury (TBI). Methods Thirty-six clean adult male SD rats were randomly divided into NT group (normal temperature), MHT 15 min group, MHT 2 h group and MHT 4 h group. TBI rat model was established by electronical controlled cortical injury device. The rats in the NT group were treated with normothermia (37℃) and the rats in the three hypothermia groups were implemented with low temperature (33.0±1.0)℃at 15 min, 2 h and 4 h for 6 h respectively after establishment of TBI model. The modified neurological senerity scores (mNSS), morphological changes in hippocampal CA1 areas, immunohistochemical staining and Western blot assay for Bcl-2, Bax and Caspase-3 were compared 3 days after TBI between the four groups. Results The neurological behavioral deficits were found in each group. Compared with the NT group, the mNSS were decreased in the three hypothermia groups (P<0.01). The results of HE staining showed that the structure of neurons was regular and arranged neatly, and the number of neurons decreased with alleviated nuclear fragmentation and dissolution in hypothermia groups. Compared with the NT group, the expression of Bcl-2 was upregulated, and the expressions of Bax and Caspase-3 were downregulated in three hypothermia groups (P<0.05). The above experimental results were superior in MHT15 min group to MHT 2 h group, and the therapeutic effect in MHT 2 h group was similar to MHT 4 h group. Conclusion The proper delayed mild hypothermia treatment could inhibit neuronal apoptosis and alleviate brain damage.
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Objective@#To analyze the prognostic significance of TP53, Bcl-2, Bcl-6, Myc proteins expression by immunohistochemical method (IHC) in diffuse large B cell Lymphoma (DLBCL) .@*Methods@#Clinical and pathologic data of 223 patients with DLBCL hospitalized in Zhejiang First Hospital from March 2009 to June 2015 were retrospectively analyzed.@*Results@#The 223 cases, a median age of 56 years old with a male predominance, had shown a 39.0% of TP53 positive expression, 38.6% of Myc, 69.1% of Bcl-2, 56.5% of Bcl-6, and 22.7% of Myc/Bcl-2 double expression. According to Hans’ classification, 27.4% were GCB and 72.6% were non-GCB. With a median follow-up of 38 (2-97) months, the 3 and 5 years survival rates were 70% and 66% , respectively. By multivariate analysis, TP53 over-expression and Myc/Bcl-2 double expression were independently associated with poor outcomes. 3-year and 5-year overall survival were 59% and 57% for patients with TP53 positive, 77% and 71% for patients with TP53 negative expression. Patients with non-GCB subtype receiving chemotherapy combined with rituximab had a higher OS than those without rituximab. But rituximab did not improve the prognosis of patients with TP53 positive.@*Conclusion@#Myc/Bcl-2 double expression and TP53 over-expression are poor prognosis for DLBCL patients. Patients with Myc/Bcl-2 double expression have shorter OS. Patients with non-GCB subtype who received chemotherapy combined with rituximab have a better OS than those without rituximab. But rituximab does not improve the prognosis of patients with TP53 positive.
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Objective To investigate the effects of sinomenine on hind limb ischemia?reperfusion ( I∕R) injury and expression of Bcl?2 and Bax in skeletal muscle cells of rats. Methods Fifty?four healthy adult male Wistar rats, aged 6-8 weeks, weighing 180-220 g, were divided into 3 groups ( n=18 each) using a random number table: sham operation group ( group S) , group I∕R and sinomenine group ( group SIN) . The rats were subjected to 4 h of ischemia on the proximal part of the right hind limb using elastic rubber bands followed by reperfusion in I∕R and SIN groups. Sinomenine 60 mg∕kg was injected intraperito?neally at 30 min before reperfusion in group SIN, and the equal volume of normal saline was given instead of sinomenine at 30 min before reperfusion in S and I∕R groups. Immediately after onset of reperfusion and at 4 and 24 h of reperfusion, blood samples were collected from the cardiac apex to measure the concentra?tions of serum lactate dehydrogenase ( LDH) and creatine kinase ( CK) . The animals were sacrificed imme?diately after blood sampling, and the gastrocnemius specimens of the hind limb were immediately removed for determination of the wet to dry weight ratio ( W∕D ratio) and expression of Bcl?2 and Bax in gastrocnemi?us cells ( by immunohistochemistry) and for examination of the pathological changes after haematoxylin and eosin staining. The Bcl?2∕Bax ratio was calculated. Results Compared with group S, the gastrocnemius W∕D ratio and concentrations of serum LDH and CK were significantly increased, the expression of Bcl?2 was significantly down?regulated, the expression of Bax was significantly up?regulated, and the Bcl?2∕Bax ratio was significantly decreased in I∕R and SIN groups ( P<0?05) . Compared with group I∕R, the gastroc?nemius W∕D ratio and concentrations of serum LDH and CK were significantly decreased, the expression of Bcl?2 was significantly up?regulated, the expression of Bax was significantly down?regulated, and the Bcl?2∕Bax ratio was significantly increased in group SIN ( P<0?05) . The pathological changes of the gastrocne?mius were significantly attenuated in group SIN as compared with group I∕R. Conclusion Sinomenine can attenuate hind limb I∕R injury, and the mechanism may be related to maintenance of the balance between Bcl?2 and Bax and to inhibition of apoptosis in skeletal muscle cells of rats.
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Objective To investigate the mechanisms of radiotherapy sensitization role of curcumin in cervical carcinoma Hela cells.Methods Western blot was used to detect expressions of p53, p21, Bax,and Bcl-2 proteins after cervical cancer Hela cells were treated.Results The expressions of p53 and p21 proteins were increased.The ratio of Bcl-2/Bax was decreased when curcumin and irradiation were applied to cells alone (P ＜ 0.05).This change were much more obvious when irradiation and curcumin were combined than either of them was used alone(P ＜0.01).Conclusions The mechanisms of curcumin enhancing radiosensitivity on Hela cells might be due to upregulation of p53, p21, and Bax proteins and downregulation of Bcl-2 protein.
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Objective To explore the effect of black garlic extract on HeLa cells and its possible molecular mechanisms.Methods Inhibitory rate of HeLa cells was detected with methyl thiazolyl tetrazolium(MTT).Flow cytometry annexin V-fluorescein isothiocyanate/propidium iodide (V-FITC/PI) was used to detect tumor cell apoptosis.Flow cytometry was used to measure cell-cycle changes.Immunohistochemistry method was used to detect expressions of tumor proteins bax and Bcl-2.Results Black garlic extract significantly inhibited the growth of HeLa cells.Black garlic extract significantly induced apoptosis of HeLa cells.The apoptosis rate was (53.26 ± 1.78)％ in the black garlic high-dose group,and (3.68 ±0.11)％ in the control group.Black garlic extract affected cell cycle,upregulated bax expression,and downregulated bcl-2 expression.Conclusions Black garlic extract significantly induced the apoptosis of HeLa cells.This effect might be caused through increasing G2/M cells or changing expressions of bax and bcl-2.
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Objective To investigate the expressions and significances of Bcl-2 and Bax in basal-like breast carcinoma (BLBC).Methods The expressions of Bcl-2 and Bax were detected in 43 cases of BLBC, 57 cases of non-BLBC and 60 cases of normal breast tissues by immunohistochemistry,and their relationships with physiological and pathological characteristics of patients were analysized.Results The positive rate of Bcl-2 in BLBC was 69.77%,higher than 43.86% in non-BLBC (χ2 =6.647,P =0.01 0)and 21 .67% in normal breast tissues (χ2 =23.831 ,P =0.001 ).The positive rate of Bax in BLBC was 20.93%,lower than 45.61 % in non-BLBC (χ2 =6.564,P =0.01 0)and 76.67% in normal breast tissues (χ2 =31 .270,P =0.001 ).The expressions of Bcl-2 and Bax were correlated with lymphnode metastasis (χ2 =6.927,P =0.008;χ2 =6.203,P =0.01 3)and pTNMstaging of BLBC (χ2 =6.331 ,P =0.01 2;χ2 =5.972,P =0.01 5).There was negative correlation between the expression of Bcl-2 and Bax in BLBC (r = -0.408,P <0.01 0). Conclusion High expression of Bcl-2 and low expression of Bax interact with each other leading to unbalance of cell deferation and apoptosis,resulting in promoting genesis and progress of BLBC.
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Objective To estimate the effect of berberine on the proliferation of and expressions of apoptosisrelated factors Bax and Bcl-2 in a human skin squamous cell carcinoma cell line A431.Methods A431 cells were cultured in vitro,and classified into various groups to be treated with berberine at different concentrations (12.5,25,5,100 mg/L) or cisplatin at 250 mg/L (positive control group) for different durations (12,24,48 and 72 hours).The A431 cells remaining untreated served as the negative control group.Subsequently,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cell growth,and inverted microscopy to observe cell morphology.Real time quantitative reverse transcription-PCR and an immunofluorescence assay were conducted to measure the mRNA and protein expressions of Bax and Bcl-2 respectively.Statistical analysis was done by multi-way analysis of variance (ANOVA) using the software SPSS 13.0.Results MTr assay showed that berberine inhibited the growth of A431 cells,and the inhibitory effect increased with the increase in concentration (F =1118.312,P ＜ 0.001) and treatment duration (F =510.927,P ＜ 0.001) of berberine.Moreover,there was a significant interaction between the concentration and treatment duration of berberine (F =70.239,P ＜ 0.001).Inverted microscopy revealed that when the concentration of berberine increased,cell density was reduced,and cell morphology changed from polygonal to round with cell body shrinkage.The ratio of bax to Bcl-2 mRNA was elevated with the increase in treatment duration and concentration of berberine,and there were significant differences in the mRNA ratio among cells treated with berberine for different time durations at same concentrations (F =226.231,1300.636,4325.139 for berberine at 25,50 and 100 mg/L respectively,all P＜ 0.001).Immunofluorescence staining indicated that the fluorescence intensity of Bax was enhanced,while that of Bcl-2 was weakened after berberine treatment.Conclusions Berberine inhibits the growth of A431 cells in a dose-and timedependent manner,and may induce the apoptosis of A431 cells via regulating the expressions of Bax and Bcl-2.
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As neoplasias mieloproliferativas crônicas cromossomo Filadélfia negativas são doenças hematológicas clonais que se caracterizam pela independência ou pela hipersensibilidade dos progenitores hematopoiéticos às citocinas. Os mecanismos celulares e moleculares envolvidos na fisiopatologia das neoplasias mieloproliferativas crônicas ainda não estão totalmente esclarecidos. Achados fisiopatológicos relevantes para as neoplasias mieloproliferativas crônicas estão associados às alterações genéticas como, por exemplo, a mutação somática no gene que codifica o JAK2 (JAK2V617F). A desregulação do processo de morte celular programada, denominada apoptose, parece participar da patogênese dessas desordens. Sabe-se que a desregulação da expressão dos genes pró- e antiapoptóticos promove a resistência das células à apoptose, culminando com o acúmulo das células mieloides e estabelecendo a neoplasia. Esta revisão enfocou as alterações na regulação da apoptose em neoplasias mieloproliferativas crônicas e a importância da melhor compreensão desse mecanismo para o desenvolvimento de novas terapias para essas doenças.
Philadelphia-chromosome negative chronic myeloproliferative neoplasms are clonal hematologic diseases characterized by hematopoietic progenitor independence from or hypersensitivity to cytokines. The cellular and molecular mechanisms involved in the pathophysiology of myeloproliferative neoplasms have not yet been fully clarified. Pathophysiologic findings relevant for myeloproliferative neoplasms are associated with genetic alterations, such as, somatic mutation in the gene that codifies JAK-2 (JAK V617F). Deregulation of the process of programmed cellular death, called apoptosis, seems to participate in the pathogenesis of these disorders. It is known that expression deregulation of pro- and anti-apoptotic genes promotes cell resistance to apoptosis, culminating with the accumulation of myeloid cells and establishing neoplasms. This review will focus on the alterations in apoptosis regulation in myeloproliferative neoplasms, and the importance of a better understanding of this mechanism for the development of new therapies for these diseases.
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Humans , Apoptosis/genetics , Mutation/genetics , Myelodysplastic-Myeloproliferative Diseases/geneticsABSTRACT
Objective To observe the effect of suramin combinated with PG-Rg3 on xenograft growth of lung adeno-carcinoma in mice, and the related mechanism thereof. Methods Forty C57BL/6J mice bearing Lewis cells were random-ized into five groups:control group, cisplatin (DDP) group, suramin group, PG-Rg3 group and combination group. Appropri-ate interventions were given in five groups of mice. Mice were sacrificed at day 24 after tumor inoculation. The subcutaneous tumors were stripped for histological examination. The tumor inhibitory rate was measured. The expressions of erythropoietin-producing hepatoma amplified sequences (Eph) B4 protein, Bcl-2 and tumors microvessel density (MVD) were determined by immunohistochemistry method with image analyze system. The apoptosis of tumor cells was measured by biotinyated dUTP nick and labeling (TUNEL) method. Results There were significantly lower values in subcutaneous tumor volume and weight in drug-treated groups than those in control group (P<0.05). The inhibitory rates were 39.20%, 49.11%, 54.86%and 62.49%in cisplatin group, suramin group, PG-Rg3 group and combined group (P<0.05). The values of EphB4, MVD and Bcl-2 grey values were significantly decreased, the apoptotic index was significantly increased, in suramin group, PG-Rg3 group and combined group than those of control group and DDP group (P<0.05). The values of EphB4, MVD and Bcl-2 grey values were significantly increased, the apoptotic index was significantly decreased, in suramin group and PG-Rg 3 group than those of combined group (P<0.05). Conclusion Suramin combinated with PG-Rg3 can produce a synergetic inhibitory activity against tumor growth of lung adenocarcinoma, which may be associated with the effect of suppressing the expression of EphB4 and angiogenesis, and the promotion of tumor cell apoptosis.
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Objective To investigate the effect of exogenous semaphorin 3A (Sema3A) on apoptosis in primary cultured rat cortical neurons and the roles of phosphoinositide 3-kinase (PI3K)/serine-threonine kinase (Akt) pathway in apoptosis induced by Sema3A.Methods Newborn Sprague-Dawley rat cortical neurons were cultured in vitro and they were identified by microtubule associated protein-2 (MAP-2) staining The cultured cortical neurons were treated with various concentrations of Sema3A (0,500,1 000,and 2 000 μg/ml) for 48hours.Neuronal survival rate was detected with CCK8 assay.Neuronal apoptosis was detected with Hoechst33342 staining and TUNEL staining.The expressions of P-Akt,Akt and Bcl-2 in cortical neurons were determined with Western blotting.Results The purity of cortical neurons culture was more than 95％.CCK8 assay showed that the survival rates of cortical neurons in the groups of 500,1 000and 2 000 μg/ml Sema3A were 80.9％ ± 5.3％,67.5％ ± 3.9％ and 50.2％ ± 4.4％ of the control group,respectively (F =165.042,P =0.000).Hoechst33342 staining showed that the apoptosis rate in the normal control group and the groups of 500,1 000and 2 000 μg/ml Sema3A were 22.4％ ± 1.2％,34.0％ ± 1.2％,39.3％ ± 1.4％ and 47.3％ ±2.3％,respectively (F =103.237,P =0.000).TUNEL staining showed that the apoptosis rate in the normal control group and the groups of 500,1 000and 2 000 μg/ml Sema3A were 23.9％ ± 1.1％,31.9％ ± 1.0％,40.1％ ± 1.5％ and 51.4％ ± 3.4％,respectively (F =103.118,P =0.000).Western blotting showed that the expressions of P-Akt (F =15.959,P =0.001) and Bcl-2 (F=18.776,P =0.001) decreased gradually,while the expression of Akt had no significant changes (F =0.590,P =0.639).Conclusions Sema3A can decrease the survival rate of the cultured cortical neurons,mainly by inducing apoptosis,and the mechanism of which might be related to the down-regulation of expressions of P-Akt and Bcl-2.
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Objective To investigate the expressions of signal transducer and activator of transcription factor 3 (STAT3),Bcl-2,and matrix metalloproteinase 2 (MMP2) in colorectal adenomas and adenocarcinomas,and the relationship of those factors with the colorectal clinicopathological features and prognosis of intestinal adenocarcinomas,and explore their roles in invasion,metastasis,and prognosis of a colorectal cancer.Methods The samples were selected from Dongyang City People's Hospital from January 2011 to January 2012,including 50 cases of paraffin-coded colorectal mucosas with chronic inflammation,50 cases of paraffincoded colorectal adenomas,and 100 cases of paraffin-coded colorectal adenocarcinomas (35 cases with metastasis and 65 cases without distant metastasis).Envison two method was used to detect the expressions of STAT3,Bcl-2,and MMP2 in each sample.Results The expressions of STAT3,Bcl-2,and MMP2 in colorectal adenomas and adenocarcinomas were significantly higher than those in colorectal mucosas with chronic inflammation (P ＜ 0.05).The expressions of STAT3 and MMP2 in colorectal adenocarcinomas with distant metastasis were significantly higher than that those without distant metastasis (P ＜ 0.05).The expression of BCL-2 had no significant difference between colorectal adenocarcinomas with and without distant metastasis (P ＞ 0.05).Conclusions STAT3,Bcl-2 and MMP2 were associated with occurrence and development of colorectal carcinoma.STAT3 was associated with distant metastasis of colorectal carcinoma and might indicate a poor prognosis.STAT3 might be used as a candidate clinical sign for recurrence,metastasis,and poor survival prognosis of colorectal adenocarcinoma.
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Objective To observe the expressions of autophagy-related proteins Beclin-1and apoptosis-related pro-teins Bcl-2 in CA1 area of hippocampus in vascular dementia model rats. Methods The experimental rats were randomly divided into sham operation group and vascular dementia model group. The model group was divided into five subgroups:1, 2, 4, 8 and 12-w group (n=6 for each group). The vascular dementia rat model was established by blocking four vessels. Learning and memory abilities were detected by Morris water maze. The expressions of Beclin-1 and Bcl-2 were detected by immunohistochemistry assay. Results The positive expressions of Beclin-1 and Bcl-2 were detected at 1-w in CA1 area of hippocampus in model rats, which reached the peak at 4-w and began to decline in 8-w, maintained the higher levels at 12-w. There were significantly higher expressions of Beclin-1 and Bcl-2 at all-time points in model group than those of sham operational group (P＜0.05 or P＜0.01). The positive expression of Beclin-1 was positively correlated with Bcl-2 at all-time points in CA1 area of hippocampus in model group (r=0.809, P＜0.05). Conclusion Results showed that autophagy and apoptosis occurred simultaneously and expressed the same trend in CA1 area of hippocampus of vascular dementia mod-el rats.
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Objective To study the role and mechanism of microRNA-16(miR-16)in the proliferation,invasion and apoptosis of ovarian epithelial carcinoma cells in vitro.Methods The SKOV-3 cells were transfected with miR-16 mimics or negative control RNA(NC)by lipofectamine 2000.The expression of miR-16 was detected by real-time reverse transcription(RT)-PCR in SKOV-3 cells,and western blot was used to detect the expression of vascular endothelial growth factor(VEGF),matrix metalloproteinase-2(MMP-2)and bcl-2 protein.Methyl thiazolyl tetrazolium(MTT),5-ethynyl-2'-deoxyuridine(EdU)and transwell assay were used to determine the proliferation and invasion abilities.And the rate of apoptotic cell was detected by flow cytometry method.Results(l)The expression level of miR16 in the transfection cells group was significantly higher than that in NC group(125.93 ± 15.30 versus 0.78 ± 0.16,P ＜ 0.01).(2)The rclative expression level of VEGF protein in transfection cells,NC and blank control group was 0.58 ± 0.05,1.22 ± 0.03,1.20 ± 0.03,MMP-2 protein was 0.63 ± 0.03,1.16 ±0.03,1.21 ± 0.03,and bel-2 protein 0.52 ± 0.03,1.19 ± 0.05,1.28 ± 0.06,respectively.The level of VEGF,MMP-2 and bcl-2 protein in the transfection group were lower than those in other control groups,and there were significantly differences among them(all P ＜0.01).(3)After transfected 4 days,the inhibition rate of cell proliferation in the transfection group was dramatically higher than that in NC group[(37.2 ±6.2)％ versus(3.6 ± 3.2)％,P =0.001].(4)The percentage rate of proliferative cells in the transfection,NC and blank control group was(12.3 ± 0.8)％,(23.4 ± 1.8)％,(31.1 ± 4.9)％.And it was lower in the transfection group(P ＜ 0.05).(5)Decreased cells via the transwell member in the transfection group(6 ± 3)were detected as compared with NC group(40 ± 9)and blank control group (48 ± 8,P ＜ 0.01).(6)Twenty-four hours after cultured in serum starvation and hypoxia,the rate of the viable and late apoptotic cells in the transfection group were significantly higher than those in NC group and blank control group[the rate of viable apoptotic cell was(16.9 ± 2.1)％,(10.3 ± 1.7)％ and(9.0 ±0.8)％ respectivcly,P＜0.01;the rate of late apoptotic cell was(13.4±3.3)％,(3.2 ±1.8)％ and (0.7 ±0.6)％ respectively,P ＜ 0.01].After cultured 48 hours,total apoptotic cells in the transfection group was significantly more than those in other groups(P ＜ 0.01).Conclusion miR-16 might inhibit the proliferation,invasion of ovarian epithelial carcinoma cells and enhance their sensitivity to apoptotic stimuli via downregulation of the expression of VEGF,MMP-2 and bcl-2 protein.
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CONTEXT: Search of tumors markers that allow treatment with higher survival rates, and indicate the response to treatment and recurrence of cancer OBJECTIVE: To analyze the immunoexpression of the proteins p53, bcl-2 and Ki-67 in colorectal adenocarcinoma and correlate them with the clinical-pathological prognostic factors. METHOD: Tissue microarray paraffin blocks were made from colorectal adenocarcinoma tissue resected from 82 patients who had undergone surgery but not chemotherapy or radiotherapy, at "Hospital São Paulo", São Paulo, SP, Brazil, between 2002 and 2005. Thin sections (4 µm) were subjected to immunohistochemical reactions, and immunoexpression staining scores were obtained. The scores were correlated with the degree of cell differentiation, staging, disease-free interval, recurrence, survival and specific mortality. The study variables were analyzed using the chi-square and Kaplan-Meier tests to investigate associations with the markers. The significance of the differences between the curves of the disease-free interval and survival was analyzed using the Logrank and Wilcoxon tests. RESULTS: The immunohistochemical expression of p53 was positive in 70 tumors (85.4 percent) and negative in 12 (14.6 percent). The expression of bcl-2 was positive in 26 (31.7 percent) and negative in 56 (68.3 percent). The expression of Ki-67 was positive in 62 (75.6 percent) and negative in 20 (24.4 percent). There was no statistically significant correlation between the expressions of these markers separately or in conjunction, in relation to the degree of cell differentiation, staging, disease-free interval, survival and specific mortality. In relation to recurrence, there was a statistically significant correlation with positive expression of Ki-67 (P = 0.035). CONCLUSION: The immunohistochemical expression of Ki-67 in colorectal cancer is associated with recurrence of this disease.
CONTEXTO: Pesquisa de marcadores tumorais que permitam tratamento com maiores índices de sobrevida, além de indicarem a resposta ao tratamento e a recurrência da neoplasia. OBJETIVO: Analisar as expressões imunoistoquímicas das proteínas p53, bcl-2 e Ki-67 no adenocarcinoma colorretal, correlacionando-as com os fatores prognósticos clínico-patológicos. MÉTODO: Foram confeccionados blocos de parafina de TMA com tecido de adenocarcinoma colorretal ressecados cirurgicamente em 82 pacientes no Hospital São Paulo da Universidade Federal der São Paulo, São Paulo, SP, de 2002 a 2005, não submetidos a radio ou quimioterapia. Cortes de 4 µm foram submetidos a reação imunoistoquímica e obtidos escores de intensidade das imunoexpressões, que foram correlacionados com o grau de diferenciação celular, estádio, tempo livre de doença, recidiva, sobrevida e mortalidade específica. As variáveis do estudo foram analisadas pelos testes do qui ao quadrado e de Kaplan-Meier para verificar as associações com os marcadores. A significância das diferenças entre as curvas do tempo livre de doença e da sobrevida foi analisada pelos testes de Logrank e Wilcoxon. RESULTADOS: A expressão imunoistoquímica da p53 foi positiva em 70 tumores (85,4 por cento) e negativa em 12 (14,6 por cento). A bcl-2 foi positiva em 26 tumores (31,7 por cento) e negativa em 56 (68,3 por cento). A expressão imunoistoquímica da Ki-67 foi positiva em 62 tumores (75,6 por cento), sendo em 20 (24,4 por cento) negativa. Não houve correlação estatisticamente significante entre as expressões imunoistoquímicas dos marcadores analisadas separadamente ou em conjunto, envolvendo o grau de diferenciação celular, estádio, tempo livre de doença, sobrevida e mortalidade específica. Com relação à recidiva, observou-se correlação estatisticamente significante com a expressão imunoistoquímica positiva da Ki-67 (P = 0,035). CONCLUSÃO: A expressão imunoistoquímica positiva da Ki-67 no câncer colorretal está...
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Adenocarcinoma/metabolism , Colorectal Neoplasms/metabolism , /metabolism , /metabolism , Biomarkers, Tumor/metabolism , /metabolism , Adenocarcinoma/pathology , Colorectal Neoplasms/pathology , Disease-Free Survival , Immunohistochemistry , Neoplasm Recurrence, Local , Neoplasm Staging , PrognosisABSTRACT
Context: Esophagogastric junction adenocarcinoma has an aggressive behavior, and TNM (UICC) staging is not always accurate enough to categorize patient's outcome. Objectives: To evaluated p53, cyclin D1 and Bcl-2 immunoexpressions in esophagogastric junction adenocarcinoma patients, without Barrett's esophagus, and to compared to clinicopathological characteristics and survival rate. Methods: Tissue sections from 75 esophagogastric junction adenocarcinomas resected from 1991 to 2003 were analyzed by immunohistochemistry for p53, cyclin D1 and Bcl-2 using streptavidin-biotin-peroxidase method. The mean follow-up time was 60 months SD = 61.5 (varying from 4 to 273 months). Results: Fifty (66.7 percent) of the tumors were intestinal type and 25 (33.3 percent) were diffuse. Vascular, lymph node and perineural infiltration were verified in 16 percent, 80 percent and 68 percent of the patients, respectively. The patients were distributed according to the TNM staging in IA in 4 (5.3 percent), IB in 10 (13.3 percent), II in 15 (20 percent), IIA in 15 (20 percent), IIIB in 15 (20 percent) and IV in 16 (21.3 percent). Immunohistochemical analysis was positive for p53, cyclin D1 and bcl-2 in 68 percent, 18.7 percent and 100 percent, respectively. There was no association between immunoexpression and vascular and/or perineural invasions, clinicopathological characteristics and patients' survival rate. Conclusion: In this selected population, there was no association between the immunomarkers, p53, cyclin D1 and bcl-2 and clinicopathological data and/or overall survival.
Contexto: O adenocarcinoma da junção esôfago-gástrica tem um comportamento agressivo e o estádio TNM não é sempre suficiente para categorizar o paciente de acordo com a evolução do mesmo. Objetivo: Avaliar a imunoexpressão do p53, ciclina D1 e Bcl-2 em pacientes com adenocarcinoma da junção esôfago-gástrica sem esôfago de Barrett e comparar com as características clínicas e sobrevida. Métodos: Cortes histológicos de 75 adenocarcinomas da esôfago-gástrica ressecados de 1991 a 2003 foram analisados por imunoistoquímica para o p53, ciclina D1 e Bcl-2, usando-se o método da estreptavidina-biotina-peroxidase. O tempo médio de seguimento foi de 60 meses, DP=61,5 (variando de 4 a 273 meses). Resultados: Cinquenta (66,7 por cento) dos tumores eram do tipo intestinal e 25 (33,3 por cento) eram difusos. Verificou-se infiltração vascular, linfonodal e perineural em 16 por cento, 80 por cento e 68 por cento dos pacientes, respectivamente. O estádio TNM foi IA em 4 (5,3 por cento), II em 15 (20 por cento), IIIA em 15 (20 por cento), IIIB em 15 (20 por cento) e IV em 16 (21,3 por cento). A análise imunoistoquímica foi positiva para p53, ciclina D1 e Bcl-2 em 68 por cento, 18,7 por cento e 100, respectivamente. Não houve associação entre a imunoexpressão e invasão vascular ou perineural, características clinicopatológicas e sobrevida geral. Conclusão: Nesta população selecionada, não houve associação entre os imunomarcadores, p53, ciclina D1 e Bcl-2 e os dados clinicopatológicos e a sobrevida geral dos pacientes.
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Cyclin D1/analysis , Esophagogastric Junction , Esophageal Neoplasms/metabolism , /analysis , Stomach Neoplasms/metabolism , /analysis , Esophageal Neoplasms/pathology , Esophageal Neoplasms/surgery , Esophagogastric Junction/pathology , Follow-Up Studies , Immunohistochemistry , Neoplasm Staging , Survival Analysis , Stomach Neoplasms/pathology , Stomach Neoplasms/surgery , Biomarkers, Tumor/analysisABSTRACT
Objective To investigate the relationship between expression of cyclooxygenase-2 (COX-2), Bcl-2 and chemosensitivities in lymph node metastases (LNMs) of gastric carcinoma. Methods The chemosenisitivities to 9 drugs were measured by MTT assay, and the expression of COX-2, Bcl-2 was determined immunohistochemically in 40 paired primary tumor (PT) and lymph node metastases(LNMs)of gastric carcinoma. Results The positive rate of COX-2 and Bcl-2 in PT were 52.5%, 45.0% respectively, and in LNMs, the positive rate were 72.5% and 60.0%. The expression of COX-2 was higher in LNMs than in PT(χ~2=4, P<0.05). There was no statistically difference in the expression of Bcl-2 between PT and LNMs(χ~2=3, P>0.05). There was positive correlation of COX-2, Bcl-2 between PT and LNMs(r=0.3403, 0.4560, beth P<0.05). There was positive correlation between COX-2 and Bcl-2 in PT and LNMs (r=0.6014, 0.5330, both P<0.01). In PT the inhibition rate for 5-FU, VCR and eADM in COX-2 high expression group were lower than those in low expression group (t=2.29, 2.18, 2.41, all P< 0.05). The inhibition rate to 5-FU, PTX and eADM was significantly lower for the Bcl-2 high expression group in PT (t=2.46, 2.23, 2.22, all P<0.05). In LNMs, there were lower inhibition rates for VCR and MTX in COX-2 strong expression group (t=2.17, 2.35, both P<0.05); the inhibition rates to 5-FU, VP-16, PTX and MTX were significantly lower for the Bcl-2 strong expression group in LNMs (t=2.32, 2.29, 2.50, 2.25, all P<0.05). Conclusions COX-2 and Bcl-2 are involved in MDR of gastric carcinoma. The LNMs of gastric carcinoma are heterogeneous with respect to expression of COX-2, Bcl-2 and response to chemosensitivities. Effective adjuvant chemotherapy in gastric carcinoma depends on targeting the metastatic component of the tumor.
ABSTRACT
Objective To investigate the possible mechanism of recombinant human erythropoietin (rhEPO) neuroprotection by studying the effect of rhEPO on expressions of matrix metalloproteinase-9 (MMP-9) and BCL-2 following focal cerebral ischemia-reperfusion in rats. Methods A rat middle cerebral artery occlusion/reperfusion (MCAO/R) model was induced by the intraluminal filament method, and intraperitoneal injection of rhEPO was used for intervention. Histopathological changes were observed by HE staining, and the expressions of MMP-9 and BCL-2 in the cerebral cortex of ischemic side were detected with immunohisto-chemistry. Results HE staining: At all time points, the numbers of surviving nerve cells were significantly higher in the rhEPO group, and their injury degree was significantly lower. MMP-9 immunohistochemistry staining: The positive cells were observed occasionally in the normal control group and the sham-operation group; the MMP-9 positive cells at the ischemic side of brain tissue in a normal saline control group began to appear at 6 hours after reperfusion, it reached the peak at 24 hours and began to decrease at 72 hours; the change trend of MMP-9 positive cells in the rhEPO group was similar to that in the normal saline control group, but it was significantly lower than that in the normal saline control group at the same time points (t were 12. 023 6, 12. 635 0, 12. 779 6, respectively, all P <0. 01). BCL-2 immunohistochemistry staining: No positive cells were found in the normal control group and sham-operation group. The numbers of BCL-2 positive cells reached the peak at the ischemic side of brain tissue in the normal saline control group at 6 hours after reperfusion, it reached the peak at 24 hours and further decreased at 72 hours; the change trend of BCL-2 positive cells in the rhEPO group was similar to that in the normal saline control group, but it was significantly higher than that in the normal saline control group at the same time points (t were 5. 763 1,8. 110 1, and 5. 798 7, respectively, all P <0. 01). Conclusions rhEPO may inhibit cortical neuronal apop-tosis at the ischemic side by inhibiting MMP-9 expression and up-regulating BCL-2 expression so as to play a neuroprotective effect.